Coexpression and Heteromerization of Two Neuronal K-Cl Cotransporter Isoforms in Neonatal Brain

Abstract

The neuron-specific K-Cl cotransporter KCC2 maintains the low intracellular chloride concentration required for the fast hyperpolarizing actions of inhibitory neurotransmitters. The KCC2 gene codes for two isoforms, KCC2a and KCC2b, which differ in their N termini. The relative expression and cellular distribution of the two KCC2 protein isoforms are unknown. Here, we characterize an antibody against the KCC2a isoform and show that a previously described antibody against KCC2 is specific for the KCC2b isoform (Hubner, C. A., Stein, V., Hermans-Borgmeyer, I., Meyer, T., Ballanyi, K., and Jentsch, T. J.

Neto2 is a KCC2 interacting protein required for neuronal Cl − regulation in hippocampal neurons

“…We determined biochemically that Neto2 preferentially interacts with oligomerized KCC2. KCC2 oligomerization, which depends on tyrosine phosphorylation of the C terminus (29), correlates with the maturation of inhibitory neurotransmission in the CNS (28), and is believed to play a major role in the change from the inactive to the active state of this transporter (5,30). Next, we demonstrated electrophysiologically that Neto2-null neurons were unable to extrude a Cl − load, indicating that the function of the remaining neuronal KCC2 in Neto2-null is severely compromised.…”

“…Efficient KCC2-mediated Cl − extrusion occurs when KCC2 exists in the oligomerized state (28)(29)(30). We, therefore, tested whether Neto2 could associate with the active oligomeric fraction of KCC2.…”

Early Growth Response 4 Mediates BDNF Induction of Potassium Chloride Cotransporter 2 Transcription

“…During development, expression levels of KCC2a mRNA in hippocampus are relatively constant, whereas KCC2b mRNA increases significantly. This indicates that the developmental increase in the expression of KCC2 mRNA and protein is mainly due to the KCC2b isoform (Uvarov et al, 2007(Uvarov et al, , 2009.…”

A Single Seizure Episode Leads to Rapid Functional Activation of KCC2 in the Neonatal Rat Hippocampus

“…Incubation with the respective antiserum diluted in TBST/milk was performed overnight in the refrigerator with agitation. Rabbit-anti-panKCC2 (1:1000; raised against amino acid residues 929-1045 of rat KCC2) (Ludwig et al, 2003) was used for KCC2; rabbit-anti-KCC2a (1:1000; raised against amino acid residues 20-40) (DPESRRHSVADPRRLPREDVK) of rat KCC2a (Uvarov et al, 2009); rabbit-anti-KCC2b (1:1000; raised against amino acid residues 8-22) (CEDGDGGANPGDGNP) of rat KCC2b (Hübner et al, 2001); rabbitanti-Na-K-ATPase ␣2 (1:1000; Millipore, catalog #AB9094); rabbit-antitubulin (1:20,000; Nordic BioSite, catalog #PRB-435P) for the neuronal class III ␤-tubulin; and mouse-anti-transferrin receptor (TfR) (1:200; Zymed, catalog #13-6800) for the transferrin receptor. An affinitypurified version of the anti-panKCC2 antibody (1:5000) was used in some experiments.…”

“…After three washes in TBST, bound antibodies were detected using an enhanced chemiluminescence kit (Pierce) and an LAS-3000 documentation system (Fujifilm). The conditions used ensured that KCC2 immunoreactivity was mainly present as a band with a molecular weight of ϳ135 kDa (Blaesse et al, 2006;Uvarov et al, 2009). Quantification of the chemoluminescence signals was performed by Advanced Image Data Analysis imaging software (Raytest).…”